In a liquid chromatography (LC) system, a mobile phase consisting of one or more solvents is driven under high system pressure through a separation unit, which often is provided in the form of a chromatography column. In high-performance LC (HPLC) systems and ultra high-performance LC (UHPLC) systems, the system pressure may be as high as, for example, about 1200 bar. The column contains a stationary phase, which in LC is typically provided in the form of a packed bed of particles such as, for example, silica beads. The particles are formulated and/or functionalized so as to separate different analytes or components (e.g., chemical compounds) in a sample. As the sample flows through the column, the sample contacts the stationary phase. The different components of the sample have different affinities for the stationary phase. This causes the different components to separate from each other as the liquid flows through the column. Consequently, the different components elute from the column outlet at different times. Hence, the output of liquid from the column contains a series of bands, each band consisting of a distinct component of the sample. That is, the bands respectively consist of the different components of the sample that were separated from each other by the column.
From the column outlet, the mobile phase and the series of bands carried therein flow to a detector configured to detect each individual band. As one example, the detector may include a flow cell through which the liquid flows, a light source, and a light detector configured to make optical-based measurements (e.g., absorbance) on the liquid flowing through the flow cell. Electrical signals produced by the detector may then be utilized to produce a chromatogram. Typically, the chromatogram plots signal intensity as a function of retention time, or alternatively as a function of retention volume. The data plot appears as a series of peaks corresponding to the series of respective bands detected by the detector. In analytical chromatography, the chromatogram is utilized to identify components in the sample and indicate their relative concentrations in the sample. Alternatively, in preparative chromatography the separating power of the column may be utilized to purify the sample, for example to isolate a target compound from other compounds contained in the sample.
An LC system may be coupled to a mass spectrometry (MS) system, thereby forming a hybrid LC-MS system. A mass spectrometry (MS) system in general includes an ion source for ionizing components of a sample under investigation, a mass analyzer for separating the gas-phase ions based on their differing mass-to-charge ratios (or m/z ratios, or more simply “masses”), an ion detector for counting the separated ions, and electronics for processing output signals from the ion detector as needed to produce a user-interpretable mass spectrum. Typically, the mass spectrum is a series of peaks indicative of the relative abundances of detected ions (e.g., ion signal intensity such as number of ion counts for each ion detected) as a function of their m/z ratios. In a hybrid LC-MS system, the separated compounds eluting from the LC column are introduced into the ion source of the MS system. The compounds are ionized in the ion source, and the resulting ions are transferred into the mass analyzer and detected on a mass-discriminated basis. A hybrid LC-MS system is thus capable of acquiring three-dimensional (3D) LC-MS data from a sample, characterized by elution time (or acquisition time or retention time), ion abundance, and m/z ratio as sorted by the MS.
Historically LC was performed with an organic solvent and a polar stationary phase, such as silica or alumina. With this “normal phase chromatography” more polar analytes bind to the polar stationary phase. Those analytes are retained by the column and elute later. Non-polar analytes bind less strongly and elute earlier from the column. Beginning in the 1980's reversed-phase chromatography (RPC) became widely used. In RPC an aqueous or polar solvent is used and the silica is functionalized with hydrophobic groups, e.g., octadecyl chains (C-18). In RPC the order of elution is the opposite or reversed. A non-polar analyte will be retained longer by the non-polar stationary phase and elute later. Conversely, the more polar analytes do not bind to the functionalized silica and elute earlier in the polar mobile phase.
In the life sciences one problem with RPC is that important polar or ionic biomolecules interact minimally with the hydrophobic column if at all and tend to elute near the column void volume. Pesek, J J, Matyska, M T, in: Wang, P G, He, H (Eds.), Hydrophilic Interaction Chromatography (HILIC) and Advanced Applications, CRC Press, Boca Raton, Fla. 2011, pp. 1-26 at 2. To overcome this issue ion-pairing techniques or hydrophilic interaction chromatography (HILIC) systems have been developed. Ion-pairing techniques coupled to RPC have been moderately successful using agents such as tetrabutylammonium hydroxide or N,N dimethylhexylamine (DMHA). Moreover, ion-pairing reagents are known to contaminate LC systems and columns that could never be washed fully away, which forces users to dedicate an LC system and column for one specific application. See Cordell et al. 2008 J Chrom B 871 115-124. On the other hand, HILIC uses a polar solvent and stationary phase that is also polar. The solvent will often contain large quantities of an organic solvent such as acetonitrile that leads to more interaction of the polar, hydrophilic analytes with the stationary phase. However, a robust HILIC column and method have not been developed to be widely accepted by the scientific community, partially due to reasons described in the next section.
Poor peak shapes are commonly observed in LC/MS experiments for highly negatively charged biomolecules such as nucleotides, or organic acids with more than one carboxylate group. See Pesek et al. 2011 J Sep Sci 34 1-8. This problem is important because good peak shape provides (1) good resolution between different analytes and (2) accurate quantitation. Using a nano-LC/MS system it was demonstrated that the poor peak shape is caused by the presence of trace metals, particularly iron, that are often found in very low concentrations from a variety of sources within the chromatographic system. See Myint et al. 2009 Anal Chem 81 7766-7772. Myint et al. used a polyamine-bonded polymer-based apHera™ NH2 column to separate organic acids and phosphorylated metabolites. They found peak shape significantly improved after flushing the column with the chelating agent, ethylenedimethyltetraamine (EDTA). However, after multiple uses of the column, the EDTA was flushed out of the system and the peak shape deteriorated. They hypothesize that the deterioration was due to metallic impurities in the organic solvent or ammonium salts. Myint et al. overcame this issue by coinjection of EDTA into the mobile phase. Myint et al. at 7769.
Other workers using a silica hydride stationary phase studied the addition of EDTA to the mobile phase to chelate metals in the system as a means to improve the chromatography. Pesek J J et al., 2011 J Sep Sci 34 3509-3516. They studied (i) flushing the system with EDTA, (ii) adding EDTA to the mobile phase, or (iii) preparing the sample with EDTA. The addition of EDTA resulted in improved peak shapes of the targeted analytes.
However, EDTA can be retained on HILIC columns and cause ion suppression for targeted compounds (see FIGS. 1A-1B and FIGS. 2A-2B herein, and Pesek J J et al., 2011). Moreover, the mass spectrometer instrument could not be used in positive ion mode with EDTA in the mobile phase due to its high ionization efficiency in positive ion mode analysis, thus rendering this approach limited to use in the negative ion mode analysis with the mass spectrometer.